Clostridium difficile is a gram-positive, spore-forming bacterium that causes a wide range of gastrointestinal disorders. Over the past decade, incidence, severity, and costs associated with C. difficile infection (CDI) have increased significantly. Difficulties in treating infections with conventional antibiotics, increasing rates f recurrent infection, and the emergence of hyper-virulent strains underscore the need for investigating new therapeutic strategies. Initiation of CDI is facilitated by disruption of the gut microbiome, most commonly mediated by broad-spectrum antimicrobial treatment, which enables C. difficile colonization and outgrowth. However, the rate of non-antibiotic associated CDI cases are well documented and steadily on the rise. This suggests that a wide range of unexplored factors likely influence susceptibility to CDI. In this proposal, we will explore the impact of the nutrient metal, Zinc (Zn), on CDI. During infection, access to nutrient metals profoundly impacts bacterial replication and virulence factor production. To exploit this, the host produces factors that limit metal availability in a process termed nutritional immunity. One such protein factor, calprotectin (CP), has strong antimicrobial properties mediated by its ability to bind Zn and manganese (Mn). Surprisingly, little work has been done to characterize the contribution of CP-mediated Zn starvation in CDI. Another factor likely impacting Zn availability during infection is diet. Altered dietary Zn levels are associated with decrease immune system function and increased susceptibility to various infections; however, there is a paucity of data on how altered dietary metal levels affect the gut microbiome. Furthermore, the impact of dietary Zn on susceptibility to CDI has yet to be defined. We hypothesize that (i) alterations in dietary metal levels profoundly impact the composition of the gut microbiome and this remodeling affects the susceptibility to C. difficile, (ii) calprotectin (CP) mediated metal sequestration is essential for limiting growth, pathogenesis, and persistence of C. difficile, and (iii) C. difficil adapts to variations in nutrient metal levels through the expression of dedicated gene systems involved in metal uptake, metabolism, and detoxification. We plan to test these three hypotheses through a series of integrated Specific Aims. First, we will define how dietary alterations in nutrient metal levels affect the murine gut microbiome and susceptibility CDI (Aim 1). We will next determine the role for CP-mediated metal starvation in CDI (Aim 2). Finally, we will identify C. difficile gene products that are required for growth and virulence in high or low n conditions (Aim 3). These experiments will elucidate the impact of altered dietary Zn levels on the gut microbiome and CDI. Furthermore, characterization of C. difficile gene products involved in nutrient metal homeostasis will lay the groundwork for future studies focused on understanding how C. difficile acquires nutrients within the host. Together, results from this proposal will lay the groundwork for the development of novel therapeutics strategies for treatment of CDI.